Small Leucine Rich Proteoglycans (SLRPs) are an expanding family of proteoglycans that contain multiple tandem repeats of a motif rich in leucine found in the extracellular matrix. The most abundant SLRP in bone is biglycan (BGN) and is, currently, the major focus of our studies. To determine the functions of BGN in vivo, transgenic mice were created that were deficient in the production of the protein (knockout/KO). These mice exhibited diminished bone mass that was progressive with age. Double tetracyline-calcein labeling revealed that the BGN deficient mice were defective in their capacity to form bone. To determine the cellular basis for the skeletal defect we isolated bone cell precursors from the marrow. Experimental analysis of the number and activity of the cells showed that the mutant mice had 1) decreased numbers of marrow stromal cells and 2) biologically compromised cells that were unable to make normal amounts of collagen mRNA and protein. When stromal cells from mutant mice were treated with TGF-beta they produced much smaller colonies than the normal animals indicating that BGN is required for proper utilization of this critical cytokine. To further explore the molecular and cellular basis for the osteopenia in the biglycan KO mice we tested the hypothesis that in addition to abnormal stromal cell function, bgn KO mice have defective osteoblasts. Cells from the calvaria of newborn mice were used because they are a rich source of homogeneous populations of osteoblasts. Cells obtained from biglycan KO mice showed a slight increase in proliferation capacity compared to normal cells suggesting a novel modulating role for biglycan in cell division. By 28 days in culture, cells from biglycan KO mice had significantly reduced levels of bone siaoloprotein and osteocalcin expression accompanied by a decrease in calcium accumulation and points to the hypothesis that the osteoblasts have a defect in their ability to differentiate. We propose that any one of these defects in osteogenic cells alone or any combination of them could contribute to the osteoporosis observed in biglycan KO mice. To test the hypothesis that functional compensation can occur between SLRPs that are from different classes we obtained mice deficient in fibromodulin. Fibromodulin was selected based on its high level of co-expression with biglycan in skeletal tissues such as the articular cartilage and differentiating osteogenic cells. At one month of age mice deficient in both fibromodulin and biglycan had decreased knee and ankle joint flexibility compared to normal mice. Radiographic and histological analysis showed that the double biglycan/fibromodulin KO mice developed ectopic ossification within the tendon resulting in the formation of supernumery sesamoid bones. By three months of age mice deficient in both fibromodulin and biglycan acquire bony cysts accompanied by severe loss of articular cartilage with exposure of the underlying bone. Preliminary transmission electron microscope analysis of patellar tendon tissue showed that collagen fibril structures are severely disturbed in the combined absence of fibromodulin and biglycan. Based on these structural observations we propose that mice deficient in biglycan and fibromodulin have functionally compromised tendons. In turn, this defect, could lead to joint instability and subsequently be the primary factor linking both ectopic ossification and premature osteoarthritis in this animal model. We are currently testing this hypothesis by comparing the biomechanical strength of isolated tendons from normal and fibromodulin/biglycan deficient mice. These studies present important new models of skeletal diseases and provide the opportunity to identify new signals that control the integrity of connective tissues. Future studies will be directed towards understanding how gene expression patterns are altered in the SLRP single and double KO mice and, further, how the responses of growth factor and hormones will be affected in vitro and in vivo.